Indian Journal of Biotechnology Vol 10, October 2011, pp 423-431

Metabolism of cyclic-di-GMP in bacterial biofilms: From a general overview to biotechnological applications

Nicoletta Castiglione 1, Valentina Stelitano 1, Serena Rinaldo 1, Giorgio Giardina 1, Manuela Caruso 1 and Francesca Cutruzzolà 1,2* 1Department of Biochemical Sciences "A. Rossi Fanelli”, Sapienza University of Rome, Rome, Italy 2Consorzio I.N.B.B., 00136 Rome, Italy

Bacteria exist in nature in a planktonic single-cell state or in a sessile multicellular state, the biofilm. In the latter state, the bacterial community optimizes the cell-environment and cell to cell communication strategies. Biofilms are widely diffuse in many industrial, environmental and clinical settings and are less sensitive to treatments with antimicrobial agents compared to planktonic cells. Biofilms formed by bacterial pathogens, such as, those formed by Pseudomonas aeruginosa in immunocompromised patients, have a high impact on public health. The switch between the planktonic and the biofilm lifestyle is strictly regulated by the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this molecule are controlled by two classes of enzymes: diguanylate cyclases (DGC) and phosphodiesterases (PDE). In this review, we report the structural and functional data available to date on these enzymes and we summarize the possible medical, environmental and industrial biotechnological applications involving bacterial c-di-GMP metabolism.

Keywords: Biofilm, cyclic-di-GMP, inhibitors, pathogenesis, PDE, DGC

Introduction The present review has focused on the human Bacteria are able to communicate and behave like a pathogen Pseudomonas aeruginosa , a well-known multicellular organism forming biofilm, a highly model organism and one of the most important organized structure consisting of cells embedded bacteria forming biofilms. Since the enzymes within a matrix of extracellular polymeric substance involved in 3',5'-cyclic diguanylic acid (c-di-GMP) (EPS) attached to a surface. As a matter of fact, synthesis and degradation are found in the bacterial cells exist in nature in a planktonic single- majority of known bacterial species 8, the information cell state or in a sessile multicellular state of the reported here is relevant for other important biofilms1. Biofilms are abundant in many industrial, pathogens. environmental and clinical settings, such as, food P. aeruginosa is an opportunistic human processing environments, potable water and medical pathogen, a leading cause of both community and devices 2,3. In particular, bacterial biofilms found on hospital acquired infections (13% of all nosocomial the surface of medical devices are a major cause of infections). P. aeruginosa biofilm is the major hospital-associated infections 4,5. Moreover, biofilms cause of death in patients of cystic fibrosis (CF), formed by pathogens play an important role in the a genetic disease affecting 1/2500 newborns in infection of living tissues and are responsible for the 9 Europe . In the CF lung, environment is poor resistance to antibiotics and to the host immune of oxygen and rich of nitrate, and under these system 2,6. Bacteria growing as microbial community conditions, P. aeruginosa is able to survive, are less sensitive to treatments with antimicrobial thanks to its anaerobic metabolism 6,10,11 , causing agents compared to planktonic cells 1,6 and produce chronic infections 10 . The bacterium is intrinsically many virulence factors 7. According to the Centers resistant to a wide array of antibiotics. Moreover, for Disease Control and Prevention (USA), 65% of it is prone to acquire new resistance genes all infections in developed countries are caused through horizontal gene transfer and produces an by biofilms. impressive array of virulence factors. The low ————— efficacy of existing therapies in eradicating *Author for correspondence: Tel: 0039-0649910713; Fax: 0039-064440062 P. aeruginosa infection calls for the development of E-mail: [email protected] new therapeutic options. 424 INDIAN J BIOTECHNOL, OCTOBER 2011

The Second Messenger 3 ′′′, 5 ′′′ Cyclic Diguanylic 5'-pGpG or GMP as products (Fig. 1). DGCs are Acid (C-di-GMP) and Its Turnover often called GGDEF proteins due to the conserved During biofilm formation, the pattern of gene amino acids found in their active site; unlikely expression is changed with respect to planktonic other bacterial domains, the GGDEF domain is cells and new intracellular signalling pathways are completely absent in eukaryotes 17 . The presence of activated. Therefore, biofilm formation can be viewed putative DGCs only in bacteria suggests that these as a developmental process 3, regulated by the key enzymes are excellent candidates for the development signal molecule c-di-GMP. This second messenger of anti-bacterial compounds. controls in bacteria an array of cellular processes Based on conserved sequence signatures, PDEs linking environmental sensing with sessile-motile are also grouped into the EAL and HD-GYP 18 transition 12,13 (Fig. 1). The clearest role for c-di-GMP families . The large number of GGDEF and EAL is, indeed, its ability to regulate the decision to switch domain proteins in a single species is somewhat 13 from a free-swimming bacterium to a surface-attached puzzling . In general, Gram-positive bacteria have bacterium 14 , determining the timing and amplitude less of DGC/PDE proteins than Gram-negative of complex processes like motility, cell adhesion, bacteria. By contrast, proteins containing the HD- biofilm formation and differentiation 15 . A low GYP domain are less common or even absent in concentration of c-di-GMP is associated with flagellar some species, whereas in other species, such as, 16 Thermotoga maritima , they account for all PDE motors or retracting pili , whereas an high amount 19,20 favours the expression of adhesins and EPS, finally activity in the cell . The number of putative DGCs leading to biofilm formation and pathogenicity 15 . and PDEs encoded in individual bacterial genomes is The exceptional importance of c-di-GMP in bacterial highly variable (for example over 29 in Escherichia physiology and pathogenesis has been recently coli , 14 in Caulobacter crescentus , 60 in Vibrio acknowledged in a commentary published in the cholerae , 41 in P. aeruginosa and none in prestigious journal Cell 14 . Helicobacter pylori ), this may reflect the ability to C-di-GMP is synthesized by the enzyme survive in different environmental niches. GGDEF diguanylate cyclase (DGC) starting from two and EAL domains are often found in tandem triphosphate (GTP) molecules and within the same protein; these hybrid proteins its degradation is mediated by specialized frequently show only one enzymatic activity with the phosphodiesterases (PDE) that yield the linear catalytically inactive domain, potentially serving a regulatory function 15 . Most DGCs and PDEs are also associated with known or hypothetical signal input domains (globin-like, PAS/PACM, GAF, HAMP, CHASE4 or membrane sensory domains MHYT or MASE1) 17 , putatively involved in sensing a range of environmental signals (oxygen, blue light, nutrient starvation, antibiotics, etc). Little is known on the intracellular receptors of c-di-GMP, which convert the increase/decrease of c-di-GMP into a biological response. Possible c-di-GMP-sensing domains include the PilZ, BcsA or PelD domains 12,21,22 , the GGDEF/EAL containing hybrid proteins 23 and even riboswitches 24 . How the DGCs and PDEs function together to produce a coherent output signal is still unclear; different c-di-GMP circuits could be separate in time and in space through 15 compartmentalization . Fig. 1—Cyclic-di-GMP turnover: Cyclic-di-GMP synthesis and degradation are, respectively, controlled by two classes of In the P. aeruginosa , PAO1 genome presents 41 enzymes, diguanylate cyclases (DGC), characterized by a GGDEF ORFs containing putative DCG (GGDEF) and/or PDE domain, and phosphodiesterases (PDE), characterized by an EAL (EAL or HD-GYP) genes (Table 1). Insertional mutants or a HD-GYP domain. Cyclic-di-GMP controls many cellular processes like motility, virulence, biofilm formation and in the DGC or DGC-PDE genes show an effect, either 25,26 differentiation 12 . reducing or increasing biofilm formation . CASTIGLIONE et al : CYCLIC-DI-GMP METABOLISM AND BIOFILM TREATMENT 425

Table 1—Genes involved in c-di-GMP turnover in P. aeruginosa PAO1 PAO1 Predicted domains Size (AA) PAO1 Predicted domains Size code code (AA)

DGC (GGDEF domain: 2GTP --> c-di-GMP) PA0169 GGDEF 235 PA3177 GGDEF 307 PA0290 PAC-GGDEF 323 PA3343 TM-TM-TM-TM-TM-GGDEF 389 PA0338 PAC-GGDEF 376 PA3702 ResponseReg.-GGDEF 347 (WspR)

PA0847 TM-CHASE4-TM- HAMP-PAS-PAC- 735 PA4332 TM-TM-TM-TM-TM-GGDEF 487 GGDEF PA1107 TM-TM-TM-TM-TM-GGDEF 398 PA4396 ResponseReg. -GGDEF 366 PA1120 TM- HAMP-GGDEF 435 PA4843 ResponseReg .-GGDEF 542 (TpbB) PA1851 TM-TM-TM-TM-TM-GGDEF 401 PA4929 7TMR-DISMED2-GGDEF 680 PA2771 GAF-GGDEF 341 PA5487 GGDEF 671 PA2870 GGDEF 525

Hybrid proteins (GGDEF + EAL domains) PA0285 TM-TM-PAS-PAC-PAS-PAC-GGDEF- 760 PA3258 EAL-CBS-GGDEF 601 EAL PA0575 TM-SPB_BAC_3-TM-PAS-PAC-PAS- 1245 PA3311 TM- MHYT- MHYT- MHYT- 783 PAC-PAS-PAC-PAS-PAC-GGDEF-EAL GGDEF-EAL PA0861 TM-TM-PAS-GGDEF-EAL 818 PA4367 TM-TM- GGDEF-EAL 687 (BifA) PA1181 MASE1-PAS-PAC-PAS-PAC-GGDEF- 1120 PA4601 TM-TM-PAS-PAC-PAS-PAC-PAS- 1415 EAL (MorA) PAC-GGDEF-EAL PA1433 TM- HAMP-GGDEF-EAL 650 PA4959 ResponseReg .- GGDEF-EAL 691 (FimX) PA1727 TM-TM- MHYT- MHYT- MHYT- 685 PA5017 TM-GAF-PAS-PAC-GGDEF-EAL 899 (MucR) GGDEF-EAL PA2072 TM-CHASE4-TM-PAS-GGDEF-EAL 864 PA5295 GGDEF-EAL 558 PA2567 GAF-GGDEF-EAL 587 PA5442 TM-TM-TM-TM-TM-PAS-PAC-PAS- 951 PAC-GGDEF-EAL

PDE (EAL domain: c-di-GMP --> pGpG) PA2133 EAL 285 PA3825 TM-EAL 526 PA2200 EAL 531 PA3947 ResponseReg .-EAL 392 (RocR) PA2818(Arr) TM-TM-EAL 525

PDE (HD-GYP domain: c-di-GMP --> 2GMP) PA4108 HD-GYP 414 PA2572 ResponseReg-NY-GYP (degenerated) 447 PA4781 ResponseReg -HD-GYP 393 GGDEF = Diguanylate cyclase, EAL = Diguanylate phosphodiesterase, HD-GYP = Metal dependent phosphohydrolases, TM = Trans membrane helix, PAS = Signal sensor domain, PAC = Signal sensor domain, GAF = cGMP-dependent 3',5'-cyclic phosphodiesterase, ResponseReg = Response regulator receiver domain, MHYT = Bacterial signalling protein N-terminal repeat, HAMP = Linker domain, CBS = Predicted adenosyl sensory domain, CHASE4 = Extracellular sensory domain, MASE1 = Predicted integral membrane sensory domain, SPB_BAC_3 = Bacterial extracellular solute-binding proteins (family 3), 7TMR-DISMED2 = T wo distinct types of extracellular sensing domains 426 INDIAN J BIOTECHNOL, OCTOBER 2011

To date, few biochemical data are available on this domain contains the Mg 2+ ion (necessary for the proteins involved in c-di-GMP turnover. A brief catalysis) and it is connected via a helical stalk summary of the major structural and functional to the GGDEF domain; the latter is oriented in such data on the DGCs and the PDEs is given below. a manner that the two active sites face each other, similar to the active conformation of adenylate Diguanylate Cyclases cyclases 31 . In addition, binding of a c-di-GMP The structure of the GGDEF domain resembles that dimer to the conserved inhibitory site (I-site) is of the adenylate cyclase catalytic domain, as evident observed 32 (Fig. 2). In summary, the protein in the structure of the PleD response regulator from is activated by phosphorylation and inhibited by 27 Caulobacter crescentus , which is considered as a product binding to the I-site; however, the feedback prototype DGC. PleD is composed of two CheY-like inhibition mechanism of WspR is even more phosphoryl receiver domains and a GGDEF domain. complex than in PleD 33 . Activation is achieved by phosphorylation of an aspartate residue that induces dimerization: two GTP Phosphodiesterases of EAL Subtype molecules, bound to the active sites of the GGDEF The crystal structures of few proteins with domains, come together to form c-di-GMP. PleD is EAL domains, such as, TdEAL from Thiobacillus regulated by non-competitive product inhibition: c-di- denitrificans , YkuI from Bacillus subtilis 34 and GMP can bind to a high affinity inhibition site (I-site) BlrP1 from Klebsiella pneumoniae 35 , have been causing a conformational change that separates the 28 determined. The general fold of the EAL domain two GGDEF domains, thus hampering catalysis . consists of a β-barrel harbouring the catalytic residues In P. aeruginosa , the only known DGC structure at the top of the barrel (Fig. 3). is that of WspR 29 (Fig. 2). This DGC has a In the case of P. aeruginosa, the catalytic similar domain organization as PleD, but lacks mechanism of the RocR protein has been the second CheY-like domain. As observed with 36 discussed and its structural model has been settled ; PleD, WspR appears to be regulated by very recently, crystallization and a preliminary phosphorylation of the N-terminal CheY-like diffraction analysis of this protein has been domain 30 . In the structure of the protein (Fig. 2), reported 37 . In RocR, hydrolysis of one O-3′-P ester bond to yield the linear dinucleotide 5 ′-pGpG is achieved by an activated water molecule and involves two Mg 2+ or Mn 2+ ions and seven catalytic

Fig. 2—Crystal structure of WspR from P. aeruginosa : The Fig. 3—Crystal structure of FimX EAL domain from N-terminal CheY-like phospho-receiver domain is connected via a P. aeruginosa : The domain possesses 11 α-helices and 8 β- helical stalk to the GGDEF domain with diguanylate cyclase strands, and its fold resembles a β-barrel. Cyclic-di-GMP activity. Cyclic-di-GMP molecules bound to the inhibitory site molecules bound to the domain are shown as black sticks. (I-site) are located distal to the active site and are shown as black The region connecting the EAL domain and the GGDEF one (the sticks. Mg 2+ ions are shown as black spheres 31 (Protein Data Bank latter not shown in this figure) forms a helix (shown in black) 38 id. 3BRE). (Protein Data Bank id. 3HV8). CASTIGLIONE et al : CYCLIC-DI-GMP METABOLISM AND BIOFILM TREATMENT 427

residues, including the Glu residue of the EAL hydrolysis and it hasa cryptic negative influence signature motif. Moreover, the structural model on swarming. It is not excluded that the YN-GYP of RocR revealed that the amino acids of the domain interferes with the action of the DFG(T/A)GYSS motif form a loop (loop 6), which HD-GYP domain proteins PA4108 and PA4781 26 . connects elements of secondary structure, a β-sheet Nevertheless, all three proteins regulate virulence and an α-helix. This motif seems to play an of P. aeruginosa , since their mutation led to a important role in signal transduction. The sequence reduction of the bacterial virulence in the larvae analysis of the 5,862 EAL domains in the bacterial of Galleria mellonella 26 . HD-GYP domain proteins genomes revealed that about half of the EAL have also been found in a number of other domains harbour a degenerated loop 6, suggesting bacterial pathogens of animals, including that the mutation of this loop may indicate a Clostridium perfringens, Bordetella bronchiseptica divergence of function for EAL domains during and Treponema denticola. For their importance evolution 38 . in virulence and since the biochemical and The absence of the essential catalytic residues structural information about them are still poor, a can serve as markers for identifying catalytically thorough characterization of this class of proteins inactive EAL domains in the bacterial genomes 36 . is highly desirable. Based on the conservation of the signature sequence, EAL domains can be divided into 3 Hybrid Proteins Containing Both GGDEF and subgroups. The EAL domains belonging to class 1 EAL Domains possess conserved catalytic residues and a conserved As previously mentioned, the hybrid proteins loop 6 and function as true PDEs. The class 2 displaying both DGC and PDE domains may act as EAL domains contain conserved catalytic residues enzymes or may be c-di-GMP sensors. Given the high and a degenerated loop 6: these EALs, as for complexity of their domain architecture, obtaining example YkuI of B. subtilis , are most likely structural data for this class of hybrid proteins is catalytically inactive, but might be activated of crucial importance to infer their function. by terminal signalling domains. The class 3 EAL Structural and functional studies of LapD, an domains lack one or more of the catalytic residues internal membrane protein that regulates surface and have a degenerated loop 6: these EALs attachment in P. fluorescens , and of FimX from are predicted to be catalytically inactive. It has P. aeruginosa 40 indicate that both proteins function been suggested that this classification also allows as c-di-GMP-sensors, which are able to communicate to discriminate whether a putative EAL protein the levels of c-di-GMP from cytoplasm to has phosphodiesterase activity or is a c-di-GMP periplasm 23 . The overall fold of EAL domain of sensing domain 39 . FimX (Fig. 3) 40 is very similar to that of the putative EAL domain-containing phosphodiesterases TdEAL Phosphodiesterases of HD-GYP Subtype from T. denitrificans and YkuI from B. subtilis, No structure is available to date for PDEs described above. On the other hand, the structure containing the HD-GYP domain. This domain is of the GGDEF domain of FimX shows an overall widespread in bacteria (over 1000 genes have similar fold to that of PleD and WspR, but lacks been found). It is classified as a metal-dependent the characteristic primary site motif of the I-site. phosphohydrolase and a divalent cation (most likely binding studies in solution confirmed that Mg 2+ or Mn 2+ ) is required for catalysis, but the EAL domain works as the sole c-di-GMP binding molecular mechanism of action is still unknown. module of FimX 40 . In P. aeruginosa , 3 genes, viz., PA4108, It is clear from the above studies that an exhaustive PA4781 and PA2572, harbour HD-GYP domain comprehension of the molecular mechanisms (Table 1). The first two proteins have a PDE underlying c-di-GMP synthesis and breakdown is activity in vivo ; moreover, they control the swarming fundamental to conceive new approaches for motility and the production of virulence factors 26 . biofilm treatment in chronic infections, such as, On the other hand, the role of the third protein the infection of P. aeruginosa in CF patients, and PA2572, which has a different YN-GYP signature, is also for other biotechnological applications, briefly uncertain 26 ; this protein is inactive in c-di-GMP described below. 428 INDIAN J BIOTECHNOL, OCTOBER 2011

Biotechnological Applications Fluorouracil, which blocks DNA replication through

Medical Applications inhibition of nucleotide biosynthesis, also prevents As mentioned above, bacteria growing in biofilms biofilm formation at concentrations not affecting 47,48 are less sensitive to treatments with antimicrobial planktonic cell growth . agents. Hence, there is a strong need to find novel Another possibility to fight the biofilm could approaches against pathogenic bacteria. The cellular be to promote the dispersion of this multicellular processes involved in biofilm formation, maintenance community. As an example, it is known that a low and dispersal are important targets for the discovery concentration of nitric oxide (NO) in P. aeruginosa of novel chemical inhibitors 41 . In particular, the favours the biofilm dispersal, causing an increase in knowledge of the structural bases of the metabolism the sensibility of the bacterium to antimicrobial 49,11 of c-di-GMP is important to identify new targets for agents ; during this complex process, a key role is effective anti-biofilm drugs; the most ambitious played by c-di-GMP. Recent studies on P. aeruginosa objective is to find a strategy capable to interfere identified a possible molecular link between NO, c-di- selectively with the synthesis and degradation of this GMP and biofilm dispersion, but whether the molecule. response to NO occurs directly or indirectly is still not 49 Given the central role of c-di-GMP in biofilm clear . Understanding the mechanism underlying the development, compounds related in structure to c-di- NO/c-di-GMP-dependent biofilm dispersal and the GMP are expected to have inhibitory activities on signalling pathway involved can provide novel and bacterial biofilm formation 42 . C-di-GMP analogues, interesting targets for new antibacterial strategies such as, monophosphorothioic acid of c-di-GMP aimed to disperse biofilm. These strategies may (c-GpGps), cyclic bis(3'-5')guanylic/adenylic acid improve the effects of antibiotic therapy, which is (c-GpAp) and cyclic bis(3'-5')guanylic/ often insufficient against bacteria growing in biofilms. 43 (c-GpIp), have been synthesized and tested on Biotechnological Production of Vaccines the bacterial biofilm formation and on bacterial 44,45 One important aspect of c-di-GMP that has been motility . These analogues suppressed the reported in literature is the extraordinary capability formation of biofilm in the Gram-negative of this molecule to produce immunostimulation. As a P. aeruginosa as well as in Staphylococcus aureus matter of fact, this molecule has been recently in the following order: c-GpGps > cGpAp > c-Gplp. identified as a potential vaccine adjuvant for systemic The suppression of biofilm formation has also been and mucosal vaccination 50 . This application is really observed in the presence of high doses of cyclic-di- attractive because the mucosal surfaces (respiratory, GMP. This could be explained by the allosteric urogenital and gastrointestinal tracts) in humans repression exerted by this dinucleotide on its 27,29 represent an entry point for pathogens and an ideal own synthesis . It should be noted here that environment for the development of diseases; a the suppressive concentrations of cyclic-di-GMP and typical example is the P. aeruginosa lung infection in its analogues added in the culture were 100–1000 CF patients. A recent work by Yan and co-workers 51 times higher than the estimated physiological 45 demonstrated that the mucosal immune response intracellular levels of cyclic-di-GMP . Screening induced using c-di-GMP translates into protective of a commercially available library of chemical immunity against Streptococcus pneumoniae compounds with known biological activities, using colonization. a combination of three microbiological assays, has Several c-di-GMP analogues have been led to the identification of the inhibitory activity of shown to have immunostimulatory properties. A 46 sulfathiazole, a known anti-metabolite drug , towards bisphosphorothioate analogue of c-di-GMP (c-GpsGps) c-di-GMP biosynthesis. It is likely that inhibition of c- was synthesized 52 and its immunostimulatory di-GMP biosynthesis by sulfathiazole does not take properties were evaluated in vivo in comparison with place through direct inhibition of the DGC activity, c-GpGps and c-di-GMP. The results of this study but through indirect effects, such as, alteration of suggest that c-GpGps and c-GpsGps appear to the nucleotide pool, which can in turn affect the elicit the inflammatory response, even though the availability of the DGC substrate GTP. This work effect was milder than that of c-di-GMP. Despite strongly suggests that perturbation of intracellular these positive results, the molecular basis of the nucleotide pools could indeed interfere with immunostimulatory and adjuvant properties of c-di- molecular signalling leading to biofilm formation. GMP still remains a mystery. CASTIGLIONE et al : CYCLIC-DI-GMP METABOLISM AND BIOFILM TREATMENT 429

Industrial and Environmental Applications a great deal of research is still needed to use the Biofilm formation is a frequent cause of c-di-GMP as a potential vaccine adjuvant in human contamination in industrial settings. Biofilms clinical trials. are often found in the food processing To date, only a very small number of GGDEF and environment. Moreover, they have a high impact EAL domains have been characterized. For this on the deterioration of water quality. In the first reason, the main aim of the research carried out in our case, biofilm contamination during the process group is to expand this limited knowledge through may favour microbial contamination of the final structural and functional characterization of selected 3 product . In the second case, the biofilm is involved P. aeruginosa PAO1 proteins involved in c-di-GMP in the deterioration of the water quality during metabolism . This information is crucial to develop its storage and distribution, and both stages are novel molecules able to modulate the synthesis of vital importance in determining the final quality and degradation of c-di-GMP and consequently the of water. biofilm formation. The control of biofilm formation strictly depends on the effective cleaning of potential growth Acknowledgement sites. Unfortunately, the bacteria embedded into the The present work is supported by the Ministero extracellular matrix of the biofilm are often protected della Universita` of Italy (Grant No. RBRN07BMCT) from the sanitizers. and the University of Rome La Sapienza, Italy. On the other hand, the immobilization of FC acknowledges the Italian Embassy in New microorganisms in biofilms is particularly appropriate Delhi (India) for supporting the participation to the for use in environmental biotechnology processes 53 . International Conference on Genomic Sciences For example, an innovative shortcut biological (ICGS 2010) held at Madurai Kamaraj University, nitrogen removal system, consisting of an aerobic Madurai, India. submerged membrane bioreactor (MBR) and an anaerobic packed-bed biofilm reactor (PBBR), was References evaluated for treating high strength ammonium- 1 Bhinu V S, Insight into biofilm-associated microbial life, 54 J Mol Microbiol Biotechnol , 10 (2005) 15-21. bearing wastewater . 2 Bryers J D, Medical biofilms, Biotechnol Bioeng , 100 Thus, it is clear that a deep knowledge of the (2008) 1-18. processes involved in the formation or dispersion 3 Kokare C R, Chakraborty S, Khopade A N & Mahadik K R, Biofilm: Importance and applications, Indian of biofilm is important, on one side to develop J Biotechnol , 8 (2009) 159-168. new efficacious treatments for the cleaning and 4 Lindsay D & von Holy A, Bacterial biofilms within the contamination prevention of industrial plants and, on clinical setting: What healthcare professionals should the other side, to exploit the biofilm metabolism for know, J Hosp Infect , 64 (2006) 313-325. decontamination of wastewater. 5 Wenzel R P, Health care-associated infections: Major issues in the early years of the 21st century, Clin Infect Dis , 45 (Suppl 1) (2007) S85-88. Conclusions 6 Moreau-Marquis S, Stanton B A & O'Toole G A, The scientific interest in c-di-GMP metabolism Pseudomonas aeruginosa biofilm formation in the cystic drastically increased in the past few years. Being fibrosis airway, Pulm Pharmacol Ther , 21 (2008) 595-599. 7 Wagner V E, Gillis R J & Iglewski B H, Transcriptome the second messenger, it is a key player in the analysis of quorum-sensing regulation and virulence decision making between the motile planktonic factor expression in Pseudomonas aeruginosa , Vaccine , 22 and static biofilm-associated bacterial 'lifestyles'. (Suppl 1) (2004) S15-20. The metabolic network involving c-di-GMP is 8 Seshasayee A S, Fraser G M & Luscombe N M, highly complex and the exact molecular mechanism Comparative genomics of cyclic-di-GMP signalling in bacteria: Post-translational regulation and catalytic activity, of c-di-GMP action remains to be fully elucidated. Nucleic Acids Res , 38 (2010) 5970-5981. A detailed understanding of such complex 9 Driscoll J A, Brody S L & Kollef M H, The epidemiology, regulatory mechanism will not only help to explain pathogenesis and treatment of Pseudomonas aeruginosa the specificity of c-di-GMP signalling systems, infections, Drugs , 67 (2007) 351-368. 10 Hassett D J, Cuppoletti J, Trapnell B, Lymar S V, Rowe J J but may also favour a biotechnological research et al , Anaerobic metabolism and quorum sensing by aimed to develop new strategies to fight biofilm in Pseudomonas aeruginosa biofilms in chronically infected medical/industrial/environmental settings 55 . Moreover, cystic fibrosis airways: rethinking antibiotic treatment 430 INDIAN J BIOTECHNOL, OCTOBER 2011

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